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The Journal of Neuroscience, February 1, 2006, 26(5):1396-1406; doi:10.1523/JNEUROSCI.1421-05.2006
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Cellular/Molecular
The Chloride Transporter Na+-K+-Cl– Cotransporter Isoform-1 Contributes to Intracellular Chloride Increases after In Vitro Ischemia
Brooks B. Pond,1 Ken Berglund,2 Thomas Kuner,3 Guoping Feng,2 George J. Augustine,2 andRochelle D. Schwartz-Bloom1,2 1Departments of Pharmacology and Cancer Biology and 2Neurobiology, Duke University Medical Center, Durham, North Carolina 27710, and 3Department of Cell Physiology, Max-Planck-Institute for Medical Research, 69120 Heidelberg, Germany
Correspondence should be addressed to Dr. Rochelle D. Schwartz-Bloom, Department of Pharmacology and Cancer Biology, Box 3813, Duke University Medical Center, Durham, NC 27710. Email: schwa001{at}duke.edu
Ischemic episodes in the CNS cause significant disturbances in neuronal ionic homeostasis. To directly measure changes in intracellular Cl– concentration ([Cl–]i) during and after ischemia, we used Clomeleon, a novel ratiometric optical indicator for Cl–. Hippocampal slices from adult transgenic mice expressing Clomeleon in hippocampal neurons were subjected to 8 min of oxygen-glucose deprivation (OGD) (an in vitro model for ischemia) and reoxygenated in the presence of glucose. This produced mild neuronal damage 3 h later that was prevented when the extracellular [Cl–] was maintained at 10 mM during reoxygenation. OGD induced a transient decrease in fluorescence resonance energy transfer within Clomeleon, indicating an increase in [Cl–]i. During reoxygenation, there was a partial recovery in [Cl–]i, but [Cl–]i rose again 45 min later. To investigate sources of Cl– accumulation, we examined the effects of Cl– transport inhibitors on the rises in [Cl–]i during and after OGD. Bumetanide and furosemide, which inhibit Cl– influx through the Na+-K+-Cl– cotransporter isoform-1 (NKCC-1) and efflux through the K+-Cl– cotransporter isoform-2, were unable to inhibit the first rise in [Cl–]i, yet entirely prevented the secondary rise in [Cl–]i during reoxygenation. In contrast, picrotoxin, which blocks the GABA-gated Cl– channel, did not inhibit the secondary rise in [Cl–]i after OGD. [Cl–]i increases during reoxygenation were accompanied by an increase in phosphorylation of NKCC-1, an indication of increased NKCC-1 activity after OGD. We conclude that NKCC-1 plays an important role in OGD-induced Cl– accumulation and subsequent neuronal damage.
Key words: oxygen-glucose deprivation; Na-K-Cl cotransporter; Clomeleon; bumetanide; furosemide; picrotoxin
Received Dec. 18, 2004; revised Dec. 10, 2004; accepted Dec. 12, 2005.
Correspondence should be addressed to Dr. Rochelle D. Schwartz-Bloom, Department of Pharmacology and Cancer Biology, Box 3813, Duke University Medical Center, Durham, NC 27710. Email: schwa001{at}duke.edu
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